Successful pregnancy requires that the antigenically disparate fetus avoid attack by the mother's immune system. Placental trophoblast cells (TBCs) are the only blastocyst-derived cells in direct contact with maternal blood, and they play essential roles in modulating maternal immune responses to the conceptus. Interferon-3 (IFN-3) is a pro-inflammatory cytokine that plays critical roles in diverse cellular processes, including induction of apoptosis, inhibition of cell proliferation and initiation of adaptive immune responses. Importantly, IFN-3 is present in the uterus during normal pregnancy. Human TBCs from all gestational ages examined are resistant to both IFN-3-mediated apoptosis, and activation of polymorphic MHC antigen expression. This suggests that human TBCs have evolved mechanisms to evade the harmful effects of IFN-3. Responses to IFN-3 are mediated by activation of transcription through the JAK-STAT-1 pathway. Importantly, multiple pathogens evade host immune responses by disrupting the JAK-STAT pathway in response to IFN-3. The magnitude and duration of cellular responses to IFN-3 are dictated by the equilibrium between the activities of the positively acting JAKs and STATs, versus negatively acting protein tyrosine phosphatases (PTPs) and suppressors of cytokine signaling (SOCS). The PTPs that regulate IFN-3 signaling are constitutively active, but are transiently inactivated following exposure of cells to IFN-3. Inactivation of the PTPs results in maximal activation of the JAKs and propagation of the cytokine signal. We previously demonstrated that IFN-3 responses are inhibited in human primary term TBCs and TBC-derived choriocarcinoma (CC) cells by PTP-mediated repression of the JAKs. Our kinetic studies demonstrate that JAKs are inhibited in TBCs even at early time points of IFN-3 treatment. These results suggest that the PTPs are not inactivated in TBCs exposed to IFN-3. We also recently demonstrated that SOCS-1 and SOCS-3 are constitutively expressed in TBCs. Thus, in Specific Aim I, we will investigate 1) whether PTPs are differentially regulated in IFN-3-treated TBCs versus other cell types, and 2) the roles of SOCS-1 and SOCS-3 in regulating IFN-3 responses in TBCs. Our collective studies suggest that inhibition of the JAKs is the primary mechanism by which TBCs suppress IFN-3 responses, but they do not address the consequences of aberrant JAK or STAT-1 activation on trophoblastic responses to IFN-3. Recent studies identified an association between unexplained pregnancy loss and a constitutively active (CA) mutant of JAK-2. Moreover, STAT-1 plays an essential role in immunosurveillance of tumors. Thus, in Specific Aim II, we will investigate the effects of CA mutants of the JAKs and STAT-1 on TBC IFN-3 responses. It is currently unclear whether inhibition of IFN-3 signaling in TBCs occurs throughout gestation, or is restricted to specific trimesters of pregnancy. Thus, in Specific Aim III we will examine IFN-3 responses in 1st, 2nd and 3rd trimester TBCs. Our studies will provide insights into how the conceptus evades deleterious responses to IFN-3, and how aberrant IFN-3 signaling in TBCs may lead to complications of pregnancy. PUBLIC HEALTH RELEVANCE: Recent studies suggest that pregnancy is a state of controlled inflammation, and that alterations in placental levels of IFN-3 play a critical role in placental pathology versus reproductive success. Our proposed studies will shed light on how TBCs compensate for the presence of pro-inflammatory IFN-3 in the uterus during normal pregnancy and following infection by providing a comprehensive analysis of the molecular mechanism(s) by which TBCs regulate responses to IFN-3. Thus, our studies will provide insights into how the conceptus evades potentially deleterious responses to IFN-3, and how aberrant cytokine signaling in TBCs may lead to pregnancy complications. This work also has important implications for tumor immunity, for loss of responsiveness to IFN-3 by tumor cells plays an essential role in tumor immunoevasion.